A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower.

Molecular engineering seeks to create functional entities for modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most artificial molecular motors are driven by Brownian motion, in which, with few exceptions, the generated forces are non-directed and insufficient for efficient transfer to passive second-level components. Consequently, efficient chemical-fuel-driven nanoscale driver-follower systems have not yet been realized. Here we present a DNA nanomachine (70 nm × 70 nm × 12 nm) driven by the chemical energy of DNA-templated RNA-transcription-consuming nucleoside triphosphates as fuel to generate a rhythmic pulsating motion of two rigid DNA-origami arms. Furthermore, we demonstrate actuation control and the simple coupling of the active nanomachine with a passive follower, to which it then transmits its motion, forming a true driver-follower pair.

Reconfigurable Nanopolygons Made of DNA Catenanes.

The development of new types of bonds and linkages that can reversibly tune the geometry and structural features of molecules is an elusive goal in chemistry. Herein, we report the use of catenated DNA structures as nanolinkages that can reversibly switch their angle and form different kinds of polygonal nanostructures. We designed a reconfigurable catenane that can self-assemble into a triangular or hexagonal structure upon addition of programmable DNA strands that function via toehold strand-displacement. The nanomechanical and structural features of these catenated nanojoints can be applied for the construction of dynamic systems such as molecular motors with switchable functionalities.

Spin Labeling of Long RNAs Via Click Reaction and Enzymatic Ligation.

Electron paramagnetic resonance (EPR) is a spectroscopic method for investigating structures, conformational changes, and dynamics of biomacromolecules, for example, oligonucleotides. In order to be applicable, the oligonucleotide has to be labeled site-specifically with paramagnetic tags, the so-called spin labels. Here, we provide a protocol for spin labeling of long oligonucleotides with nitroxides. In the first step, a short and commercially available RNA strand is labeled with a nitroxide via a copper-(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also referred to as "click" reaction. In the second step, the labeled RNA strand is fused to another RNA sequence by means of enzymatic ligation to obtain the labeled full-length construct. The protocol is robust and has been shown experimentally to deliver high yields for RNA sequences up to 81 nucleotides, but longer strands are in principle also feasible. Moreover, it sets the path to label, for example, long riboswitches, ribozymes, and DNAzymes for coarse-grained structure determination and enables to investigate mechanistical features of these systems.

A Self-Regulating DNA Rotaxane Linear Actuator Driven by Chemical Energy.

Nature-inspired molecular machines can exert mechanical forces by controlling and varying the distance between two molecular subunits in response to different inputs. Here, we present an automated molecular linear actuator composed of T7 RNA polymerase (T7RNAP) and a DNA [2]rotaxane. A T7 promoter region and terminator sequences are introduced into the rotaxane axle to achieve automated and iterative binding and detachment of T7RNAP in a self-controlled fashion. Transcription by T7RNAP is exploited to control the release of the macrocycle from a single-stranded (ss) region in the T7 promoter to switch back and forth from a static state (hybridized macrocycle) to a dynamic state (movable macrocycle). During transcription, the T7RNAP keeps restricting the movement range on the axle available for the interlocked macrocycle and prevents its return to the promotor region. Since this range is continuously depleted as T7RNAP moves along, a directional and active movement of the macrocycle occurs. When it reaches the transcription terminator, the polymerase detaches, and the system can reset as the macrocycle moves back to hybridize again to the ss-promoter docking site. The hybridization is required for the initiation of a new transcription cycle. The rotaxane actuator runs autonomously and repeats these self-controlled cycles of transcription and movement as long as NTP-fuel is available.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism*.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

Regeneration of Burnt Bridges on a DNA Catenane Walker.

DNA walkers are molecular machines that can move with high precision onthe nanoscale due to their structural and functional programmability. Despite recent advances in the field that allow exploring different energy sources, stimuli, and mechanisms of action for these nanomachines, the continuous operation and reusability of DNA walkers remains challenging because in most cases the steps, once taken by the walker, cannot be taken again. Herein we report the path regeneration of a burnt-bridges DNA catenane walker using RNase A. This walker uses a T7RNA polymerase that produces long RNA transcripts to hybridize to the path and move forward while the RNA remains hybridized to the path and blocks it for an additional walking cycle. We show that RNA degradation triggered by RNase A restores the path and returns the walker to the initial position. RNase inhibition restarts the function of the walker.

Interlocked DNA Nanojoints for Reversible Thermal Sensing.

The ability to precisely measure and monitor temperature at high resolution at the nanoscale is an important task for better understanding the thermodynamic properties of functional entities at the nanoscale in complex systems, or at the level of a single cell. However, the development of high-resolution and robust thermal nanosensors is challenging. The design, assembly, and characterization of a group of thermal-responsive deoxyribonucleic acid (DNA) joints, consisting of two interlocked double-stranded DNA (dsDNA) rings, is described. The DNA nanojoints reversibly switch between the static and mobile state at different temperatures without a special annealing process. The temperature response range of the DNA nanojoint can be easily tuned by changing the length or the sequence of the hybridized region in its structure, and because of its interlocked structure the temperature response range of the DNA nanojoint is largely unaffected by its own concentration; this contrasts with systems that consist of separated components.

ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes.

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

Molecular Architecture of a Network of Potential Intracellular EGFR Modulators: ARNO, CaM, Phospholipids, and the Juxtamembrane Segment.

Epidermal growth factor receptors (EGFRs) are central cellular signaling interfaces whose misregulation is related to several severe diseases. Although ligand binding to the extracellular domain is the most obvious regulatory element, also intracellular factors can act as modulators of EGFR activity. The juxtamembrane (JM) segment seems to be the receptor's key interaction interface of these cytoplasmic factors. However, only a limited number of cytoplasmic EGFR modulators are known and a comprehensive understanding of their mode of action is lacking. Here, we report ARNO, a member of the cytohesin family, as another JM-binding protein and structurally characterize the ARNO-EGFR interaction interface. We reveal that its binding mode displays common features and distinct differences with JM's interaction with calmodulin and anionic phospholipids. Furthermore, we show that each interaction can be modulated by additional factors, generating a distinctly regulated network of possible EGFR modulators acting on the intracellular domain of the receptor.

Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures.

Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorporating proteins, molecular switches such as photo-switchable azobenzene derivatives, or fluorophores for studying their mechanical behavior by fluorescence quenching or fluorescent resonance energy transfer experiments. These modified interlocked DNA architectures provide access to more complex mechanical devices and nanomachines that can perform a variety of desired functions and operations. The assembly of catenanes can be completed in 2 d, and that of rotaxanes in 3 d. Addition of azobenzene functionality, fluorophores, anchor groups, or the site-specific linkage of proteins to the nanostructure can extend the time line.

Translocation of a Cell Surface Spliceosomal Complex Induces Alternative Splicing Events and Lymphoma Cell Necrosis.

Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.36. The aptamer triggers SSC internalization, causing global changes in alternative splicing patterns that eventually lead to necrotic cell death. Our study reveals an exceptional spatial arrangement of a spliceosomal complex and defines it not only as a potential target of anti-cancer drugs, but also suggests that its localization plays a fundamental role in cell survival.

Orthogonally Photocontrolled Non-Autonomous DNA Walker.

There is considerable interest in developing progressively moving devices on the nanoscale, with the aim of using them as parts of programmable therapeutics, smart materials, and nanofactories. Present here is an entirely light-induced DNA walker based on orthogonal photocontrol. Implementing two azobenzene derivatives, S-DM-Azo and DM-Azo, enabled precise coordination of strand displacement reactions that powered a biped walker and guided it along a defined track in a non-autonomous way. This unprecedented type of molecular walker design offers high precision control over the movement in back-and-forth directions as desired, and is regulated solely by the sequence of the irradiation wavelengths. This concept may open new avenues for advancing non-autonomous progressive molecular motors, ultimately facilitating their application at the nanoscale.

Temporal and Reversible Control of a DNAzyme by Orthogonal Photoswitching.

The reversible switching of catalytic systems capable of performing complex DNA  computing operations using the temporal control of two orthogonal photoswitches is described. Two distinct photoresponsive molecules have been separately incorporated into a split horseradish peroxidase-mimicking DNAzyme. We show that its catalytic function can be turned on and off reversibly upon irradiation with specific wavelengths of light. The system responds orthogonally  to a  selection of irradiation wavelengths    and   durations of irradiation. Furthermore, the DNAzyme exhibits reversible switching and retains this ability throughout multiple switching cycles. We apply our system as a light-controlled 4:2 multiplexer. Orthogonally photoswitchable DNAzyme-based catalysts as introduced here have potential use for controlling complex logical operations and for future applications in DNA nanodevices.

High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy.

Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) in the frozen state.

A bio-hybrid DNA rotor-stator nanoengine that moves along predefined tracks.

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor-stator units that consume chemical energy are elusive. Here, we report a bio-hybrid nanoengine consisting of a catalytic stator that unidirectionally rotates an interlocked DNA wheel, powered by NTP hydrolysis. The engine consists of an engineered T7 RNA polymerase (T7RNAP-ZIF) attached to a dsDNA nanoring that is catenated to a rigid rotating dsDNA wheel. The wheel motor produces long, repetitive RNA transcripts that remain attached to the engine and are used to guide its movement along predefined ssDNA tracks arranged on a DNA nanotube. The simplicity of the design renders this walking nanoengine adaptable to other biological nanoarchitectures, facilitating the construction of complex bio-hybrid structures that achieve NTP-driven locomotion.

Poly-ligand profiling differentiates trastuzumab-treated breast cancer patients according to their outcomes.

Assessing the phenotypic diversity underlying tumour progression requires the identification of variations in the respective molecular interaction networks. Here we report proof-of-concept for a platform called poly-ligand profiling (PLP) that surveys these system states and distinguishes breast cancer patients who did or did not derive benefit from trastuzumab. We perform tissue-SELEX on breast cancer specimens to enrich single-stranded DNA (ssDNA) libraries that preferentially interact with molecular components associated with the two clinical phenotypes. Testing of independent sample sets verifies the ability of PLP to classify trastuzumab-treated patients according to their clinical outcomes with ROC-AUC of 0.78. Standard HER2 testing of the same patients gives a ROC-AUC of 0.47. Kaplan-Meier analysis reveals a median increase in benefit from trastuzumab-containing treatments of 300 days for PLP-positive compared to PLP-negative patients. If prospectively validated, PLP may increase success rates in precision oncology and clinical trials, thus improving both patient care and drug development.

Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells.

Platforms for targeted drug-delivery must simultaneously exhibit serum stability, efficient directed cell internalization, and triggered drug release. Here, using lipid-mediated self-assembly of aptamers, we combine multiple structural motifs into a single nanoconstruct that targets hepatocyte growth factor receptor (cMet). The nanocarrier consists of lipidated versions of a cMet-binding aptamer and a separate lipidated GC-rich DNA hairpin motif loaded with intercalated doxorubicin. Multiple 2',6'-dimethylazobenzene moieties are incorporated into the doxorubicin-binding motif to trigger the release of the chemotherapeutics by photoisomerization. The lipidated DNA scaffolds self-assemble into spherical hybrid-nanoconstructs that specifically bind cMet. The combined features of the nanocarriers increase serum nuclease resistance, favor their import into cells presumably mediated by endocytosis, and allow selective photo-release of the chemotherapeutic into the targeted cells. cMet-expressing H1838 tumor cells specifically internalize drug-loaded nanoconstructs, and subsequent UV exposure enhances cell mortality. This modular approach thus paves the way for novel classes of powerful aptamer-based therapeutics.

Expanding the Toolbox of Photoswitches for DNA Nanotechnology Using Arylazopyrazoles.

Photoregulation is among the most promising tools for development of dynamic DNA nanosystems, due to its high spatiotemporal precision, biocompatibility, and ease of use. So far, azobenzene and its derivatives have shown high potential in photocontrolling DNA duplex hybridization by light-dependent photoisomerization. Despite many recent advances, obtaining sufficiently high photoswitching efficiency under conditions more suitable for work with DNA nanostructures are challenging. Here we introduce a pair of arylazopyrazoles as new photoswitches for efficient and reversible control of DNA hybridization achieved even at room temperature with a low number of required modifications. Their photophysical properties in the native state and in DNA strands result in near-quantitative isomerization rates by irradiation with UV and orange light. To demonstrate the applicability of these photoswitches, we have successfully applied one of them to open and close a DNA hairpin by light at room temperature.